Local structural preferences in shaping tau amyloid polymorphism

Tauopathies encompass a group of neurodegenerative disorders characterised by diverse tau amyloid fibril structures. The persistence of polymorphism across tauopathies suggests that distinct pathological conditions dictate the adopted polymorph for each disease. However, the extent to which intrinsic structural tendencies of tau amyloid cores contribute to fibril polymorphism remains uncertain. Using a combination of experimental approaches, we here identify a new amyloidogenic motif, PAM4 (Polymorphic Amyloid Motif of Repeat 4), as a significant contributor to tau polymorphism. Calculation of per-residue contributions to the stability of the fibril cores of different pathologic tau structures suggests that PAM4 plays a central role in preserving structural integrity across amyloid polymorphs. Consistent with this, cryo-EM structural analysis of fibrils formed from a synthetic PAM4 peptide shows that the sequence adopts alternative structures that closely correspond to distinct disease-associated tau strains. Furthermore, in-cell experiments revealed that PAM4 deletion hampers the cellular seeding efficiency of tau aggregates extracted from Alzheimer’s disease, corticobasal degeneration, and progressive supranuclear palsy patients, underscoring PAM4’s pivotal role in these tauopathies. Together, our results highlight the importance of the intrinsic structural propensity of amyloid core segments to determine the structure of tau in cells, and in propagating amyloid structures in disease.

In the manuscript from Louros and associates, the authors identify a new amyloidogenic motif in tau they refer to as Polymorphic Amyloid Motif of Repeat 4 (PAM4).Using a combination of approaches to characterize its role in tau assembly, the authors provide compelling data showing that the PAM4 peptide assembles into filaments that resemble disease-associated tau structures.The results are very intriguing and will significantly impact the field.However, there are a couple of issues that should be addressed prior to publication.

Response:
We thank the reviewer for the valuable comments provided.Below, we have addressed the concerns raised by the reviewer hoping to improve the clarity of our findings and the manuscript.
Point 1.The authors compare the seeding activity of different tau peptides in Figure 2i-j, but as a high purity peptide containing the PAM4 region was synthesized for characterization, it would be important to ensure all peptides included in the comparison were synthesized to a similar purity.In addition, as seeding with the PAM4 peptide was assessed following sonication of mature PAM4 peptide filaments, it is important to ensure all peptides were treated equally prior to exposure to tauRD-transfected HEK293 cells.Moreover, as the peptide 348-362 was characterized in Figure 1, while the peptide 350-362 (PAM4) was generated and examined in Figure 2, it would seem to be important to assess how the peptides 348-362 and 350-362 (PAM4) compare in the assays performed in Figure 1.In the seeding activity assay, the PAM4 peptide seems to be more effective than 348-362, but it is not clear whether this may be due to differences in purity or sonication of filaments prior to seeding.
Response to Point 1: Indeed, it is important that the above are clear to the readers.All peptide samples were prepared and treated using the same protocols prior to transfection.Briefly, as stated in the Methods section, all peptides were dissolved in parallel in the same buffer preparation (25mM HEPES, 10mM KCl, 5mM MgCl2, 3mM TCEP, 0.01% NaN3, pH 7.2) and incubated for 7 days at room temperature to form mature amyloid fibrils.The mature samples were then sonicated using a matching protocol in a Bioruptor Pico (15 min -30 sec on, 30 sec off at 10°C) and then transfected using the same transfection protocol.Therefore, the observed differences are not expected to have occurred due to treatment, as all samples were prepared in parallel under the same conditions.
Individual HPLC chromatograms and the corresponding MS analysis performed during peptide quality control are now provided as additional supplementary material indicating the purity and masses detected for every peptide synthesized and used in our work (Suppl.Figures 9-18).As seen in the provided chromatograms, all peptides were synthesized to high purities (>90%).In fact, most of the peptide hits shown in Fig. 2i-j have comparable purity levels (>94-99%) to the PAM4 peptide.Importantly, as the differences observed in seeding efficiencies were 10-to 100-fold lower compared to PAM4, it is very unlikely that such a large difference in effect is due to the small differences in purity.
During screening in Fig. 1, we showed that the 348-362 fragment forms fibrils that bind amyloid reporter dyes and have typical amyloid-like characteristics, as observed by TEM.We have now added end-state values for the kinetics shown in Fig. 2A, so both dye binding properties and the amyloid characteristics observed by TEM of fibrils formed by PAM4 are reported in Figure 2 (Fig. 2A and F).Considering the reviewer's comment, we have also incorporated a brief comparison statement for both the Th-T and TEM observations of the two peptides in the main manuscript .
Point 2. The authors use cryo-EM to demonstrate that the high purity PAM4 peptide predominantly forms Type 1 filaments, while the lower purity PAM4 peptide batch forms Type 2-4 filaments.However, as Type 2-4 filaments are more representative of disease-associated tau polymorphs, it is unclear why the lower purity peptide was not used for other assays.In addition, can the authors speculate why the high purity PAM4 peptide to form the type of filaments observed in the brain, while the lower purity peptide is able to do this?
Response to Point 2: Starting from the latter, it is very important to point out that the higher purity PAM4 batch did not fail to produce representative folds to the ones observed in the brain.In fact, it formed filaments in which PAM4 adopts the FoldB conformation, which is representative of PiD and PSP tau disease polymorphs.The aggregation assays shown in Fig. 2 were performed simply to establish the amyloidogenic properties of the PAM4 segment in isolation as an identified APR of tau.As such, to ensure that the low percentage of impurity does not affect the overall aggregation propensity of the PAM4 region, we tested the highpurity peptide, which definitively illustrates the amyloidogenic properties of the PAM4 region.
Recent work suggests that compared to an N-terminally acetylated end, an Fmoc protection group can provide additional π-π stacking interactions (Sharma R. et al. 2022 ChemBioChem 23, e202200499).Interestingly, similar interactions seem to mediate protofilament interfaces in the case of type 2-4 filaments, which could potentially explain the additional filaments recovered from the lower purity peptide batch.Prompted by the reviewer's suggestion, a brief sentence speculating on the above has been added to the main manuscript (Page 10, lines 272-277).
Point 3. Please clarify the significance of underlined sequences in Figure 1e in the figure legend; presumably these are referring to the sequences identified in Figure 1b.

Response to Point 3:
We are grateful to the reviewer for pointing this out.As correctly spotted, the underlined segments represent the sequences identified in Figure 1b.We have added this information in the corresponding figure legends (lines 917 and 937-938).